Comparison of three different plasma homocysteine assays with gas chromatography-mass spectrometry.
BACKGROUND: Various methods are available to measure plasma total homocyst(e)ine (tHcy) concentrations, but whether plasma tHcy assays may be used interchangeably is not known. METHODS: Results from three different methods [HPLC with fluorescence detection, enzyme immunoassay (EIA), and fluorescence polarization immunoassay (FPIA)] to determine fasting (n = 163) and post-methionine load (n = 80) plasma tHcy concentrations were compared with those obtained by gas chromatography-mass spectrometry (GC-MS). Difference plots on non-transformed and log-transformed data were used to assess the agreement between HPLC and GC-MS, EIA and GC-MS, and FPIA and GC-MS. RESULTS: The closest agreement between methods was observed between GC-MS and FPIA for fasting tHcy concentrations, with 95% of the FPIA values between 19% above and 24% below the corresponding GC-MS results. Post-methionine load tHcy concentrations measured by EIA showed the least agreement with GC-MS, with 95% of values measured by EIA ranging between 52% above and 16% below the GC-MS values. With respect to GC-MS, the above-mentioned methods showed a negative bias for fasting tHcy concentrations, but a positive bias for both immunoassays for post-methionine load tHcy concentrations. CONCLUSIONS: The agreement among methods is insufficient to allow them to be used interchangeably. The intermethod differences emphasize the need for standardization of plasma tHcy assays. (+info)
Comparison of plasma total homocysteine measurements in 14 laboratories: an international study.
BACKGROUND: Information on interlaboratory variation and especially on methodological differences for plasma total homocysteine is lacking. METHODS: We studied 14 laboratories that used eight different method types: HPLC with electrochemical detection (HPLC-ED); HPLC with fluorescence detection (HPLC-FD) further subdivided by type of reducing/derivatizing agent; gas chromatography/mass spectrometry (GC/MS); enzyme immunoassay (EIA); and fluorescence polarization immunoassay (FPIA). Three of these laboratories used two methods. The laboratories participated in a 2-day analysis of 46 plasma samples, 4 additional plasma samples with added homocystine, and 3 plasma quality-control (QC) pools. Results were analyzed for imprecision, recovery, and methodological differences. RESULTS: The mean among-laboratory and among-run within-laboratory imprecision (CV) was 9.3% and 5.6% for plasma samples, 8.8% and 4.9% for samples with added homocystine, and 7.6% and 4.2% for the QC pools, respectively. Difference plots showed values systematically higher than GC/MS for HPLC-ED, HPLC-FD using sodium borohydride/monobromobimane (however, for only one laboratory), and EIA, and lower values for HPLC-FD using trialkylphosphine/4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole. The two HPLC-FD methods using tris(2-carboxyethyl) phosphine/ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) or tributyl phosphine/SBD-F, and the FPIA method showed no detectable systematic difference from GC/MS. CONCLUSIONS: Among-laboratory variations within one method can exceed among-method variations. Some of the methods tested could be used interchangeably, but there is an urgent need to improve analytical imprecision and to decrease differences among methods. (+info)
Method for the determination of total homocysteine in plasma and urine by stable isotope dilution and electrospray tandem mass spectrometry.
BACKGROUND: Total homocysteine (tHcy) has emerged as an important independent risk factor for cardiovascular disease. Analytical methods are needed to accommodate the high testing volumes for tHcy and provide rapid turnaround. METHODS: We developed liquid chromatography electrospray tandem mass spectrometry (LC-MS/MS) method based on the analysis of 100 microL of either plasma or urine with homocystine-d(8) (2 nmol) added as internal standard. After sample reduction and deproteinization, the analysis was performed in the multiple reaction monitoring mode in which tHcy and Hcy-d(4) were detected through the transition from the precursor to the product ion (m/z 136 to m/z 90 and m/z 140 to m/z 94, respectively). The retention time of tHcy and Hcy-d(4) was 1.5 min in a 2.5-min analysis. RESULTS: Daily calibrations between 2.5 and 60 micromol/L exhibited consistent linearity and reproducibility. At a plasma concentration of 0.8 micromol/L, the signal-to-noise ratio for tHcy was 17:1. The regression equation for the comparison between our previous HPLC method (y) and the LC-MS/MS method (x) was y = 1.097x - 1.377 (r = 0.975; S(y|x) =1.595 micromol/L; n = 367), and for comparison between a fluorescence polarization immunoassay (Abbott IMx; y) and LC-MS/MS (x) was y = 1.039x + 0.025 (r = 0.969; S(y|x) =1.146 micromol/L; n = 367). Inter- and intraassay CVs were 2.9-5.9% and 3.6-5.3%, respectively, at mean concentrations of 3.9, 22.7, and 52.8 micromol/L. Mean recovery of tHcy was 94.2% (20 micromol/L) and 97.8% (50 micromol/L). CONCLUSIONS: The sensitivity and specificity of tandem mass spectrometry are well suited to perform high-volume analysis of tHcy. Reagents are inexpensive and sample preparation of a batch of 40 specimens is completed in less than 1 h and is amenable to automation. (+info)
Th1 polarization of the immune response in Behcet's disease: a putative pathogenetic role of interleukin-12.
OBJECTIVE: To investigate whether immunologic abnormalities in patients with Behcet's disease (BD) are related to abnormalities of the Th1/Th2 ratio. METHODS: Th1/Th2 cytokine production by peripheral blood lymphocytes (PBL) from 31 patients with BD, 11 patients with inflammatory arthritis, and 10 healthy blood donors was evaluated by intracellular immunofluorescence staining. Serum interleukin-12 (IL-12) levels were measured using an enzyme amplified-sensitivity immunoassay. The effect of recombinant IL-12 (rIL-12) on spontaneous and Fas-mediated apoptosis of phytohemagglutinin (PHA)-stimulated PBL was evaluated by flow cytometry using propidium iodide (PI) staining and a bromodeoxyuridine (BrdU)/PI procedure. RESULTS: Intracellular immunofluorescence staining of IL-2, IL-4, and interferon-gamma (IFNgamma) in CD3+ lymphocytes from BD patients demonstrated a strong polarization of the immune response toward the Th1 pathway that correlated with the progression of BD. Peripheral Th1 cells were significantly increased in patients with active disease (n = 14) as compared with those in patients in complete remission (n = 17), patients with inflammatory arthritis, and normal donors. In addition, serum IL-12 levels were correlated with peripheral Th1 lymphocytes and disease progression. Apoptotic analysis revealed that PHA-activated PBL from patients with active disease were highly sensitive to spontaneous and Fas-mediated activation-induced cell death. However, addition of rIL-12 to complete medium prevented this spontaneous and Fas-induced apoptosis and enhanced the proliferation of Th1 lymphocytes. CONCLUSION: Taken together, these results indicate that a strong Th1 immune response occurs in active BD and suggest that IL-12 plays a substantial part in the pathogenesis of BD. By preventing spontaneous and Fas-induced cell death, in fact, it results in an abnormal growth of autoreactive Th1 lymphocytes that could contribute to the prolonged inflammatory autoimmune condition of BD. (+info)
A rapid reusable fiber optic biosensor for detecting cocaine metabolites in urine.
Analyte 2000, a four-channel fiber optic biosensor (FOB), was used for analysis of cocaine and its metabolites (COC) in human urine using a competitive fluorescence immunoassay. Binding of antibenzoylecgonine monoclonal antibody (mAb) to the casein-benzoylecgonine Ag-coated, tapered optical fibers was inhibited by COC. Bound mAb, which inversely correlated with COC concentration, was quantitated by fluorescence produced by evanescent excitation of bound cyanine dye-tagged antimouse antibody (CY5-Ab). The effective concentration range of benzoylecgonine (BE) for inhibiting the fluorescent signals was 0.75-50 ng/mL, with IC50 of 9.0 ng/mL. This FOB had similar affinities for BE, cocaine, and cocaethylene, but very low affinities for ecgonine and ecgonine methyl ester. A sensitivity of 100% and a specificity of 96% were achieved when 54 human urine specimens were analyzed by FOB (cutoff, 300 ng/mL COC) and GC-MS (cutoff, 150 ng/mL BE). All results were in agreement except for one positive FOB result with a GC-MS BE concentration of 148 ng/mL. In addition, regeneration and reuse of the fiber for multiple analyses were performed successfully with no carryover from specimens containing high COC concentrations to specimens containing low COC concentrations. (+info)
Detection of methadone, LAAM, and their metabolites by methadone immunoassays.
l-Alpha-acetylmethadol (LAAM) was recently approved as a substitute for methadone. LAAM, methadone, and their common metabolite, methadol, are extensively N-demethylated. The structural similarities of LAAM and its metabolites to methadone suggest that they may cross-react in methadone immunoassays. To test this hypothesis, drug-free urine was fortified with LAAM, norLAAM, dinorLAAM, methadol, normethadol, dinormethadol, methadone, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), or 2-ethyl-5-methyl-3,3-diphenylpyrroline (EMDP) at 12 concentrations (0.03 to 100 microg/mL). Samples were analyzed using two enzyme immunoassays (Behring Diagnostics, EIA-b; Diagnostic Reagents, EIA-d); a fluorescent polarization immunoassay (Abbott, FPIA); two enzyme-linked immunosorbant immunoassays (Diagnostix, ELISA-d; STC Technologies, ELISA-s); a kinetic microparticles in solution immunoassay (Roche Diagnostic Systems, KIMS); and a radioimmunoassay (Diagnostic Products, RIA). LAAM had high cross-reactivity with ELISA-d (318.3%), RIA (249.5%), EIA-d (100.8%), KIMS (91.1%), and ELISA-s (75.3%). Methadol also displayed relatively high cross-reactivity as follows: EIA-d (97.8%), KIMS (85.4%), ELISA-d (70.3%), and FPIA (37.7%). Successive N-demethylations of LAAM and methadol were associated with loss of cross-reactivity. The methadone metabolites EDDP and EMDP showed little cross-reactivity. These findings suggest that LAAM use could result in positive immunoassay test results when using many of the commercially available methadone immunoassay kits and that confirmation of LAAM and its metabolites should be considered. (+info)
Development and evaluation of an improved method for screening of amphetamines.
We developed a homogeneous immunoassay method to eliminate false-positive amphetamine results caused by cross-reactive substances, including over-the-counter allergy and cold medications. This method uses a neutralizing antibody that binds to amphetamines but does not bind to the labeled amphetamine conjugate used in the assay. The amount of neutralizing antibody is sufficient to reduce the assay signal resulting from authentic amphetamine and methamphetamine, but not the signal resulting from cross-reactants. This concept was implemented using the CEDIA DAU Amphetamines assay on Hitachi 747 and 717 clinical chemistry analyzers. Urine samples were tested using the standard, unmodified reagents in one channel and reagents containing the neutralizing antibody in a second channel. The difference in rate between the two tests was calculated by the analyzer; true-positive samples showed a significantly greater decrease in assay signal in response to neutralizing antibody as compared with false-positive samples. The neutralization method was evaluated in two studies using 448 samples that tested positive in the initial CEDIA DAU Amphetamines screening test. The samples were separated into categories of 154 true-positive samples and 294 false-positive samples based upon a secondary screen with the Abbott FPIA Amphetamines assay followed by gas chromatography-mass spectrometry (GC-MS) testing using the HHS (SAMHSA) cutoff criteria. The CEDIA neutralization test successfully identified all 154 of the GC-MS confirmed positive samples. The test successfully identified as false positive 251 out of the 294 (85.4%) samples that failed to confirm by GC-MS. (+info)
Measurement of thiols in human plasma using liquid chromatography with precolumn derivatization and fluorescence detection.
A liquid chromatography (LC) method for the simultaneous measurement of the main low molecular mass thiols (i.e., cysteine, cysteinylglycine, homocysteine, and glutathione) in human plasma is described. The sample treatment consists of the reduction of disulfide bounds with tri-n-butylphosphine and protein precipitation with trichloroacetic acid followed by precolumn derivatization with a thiol-selective fluorogenic reagent (7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide). The structure of thiol derivatives is assessed using electrospray ionization-mass spectrometry (MS). The stability of resulting adducts in acidic medium (24 h at 10 degrees C) allows the automation of the technique and a high throughput of samples (approximately 50 per day). Separation is complete within 12 min using isocratic reversed-phase mode, and detection is operated by spectrofluorimetry (lambda ex = 385 nm and lambda em = 515 nm). Quantitation is performed by an internal standardization mode using thioglycolic acid. The LC method is fully validated, and homocysteine concentrations obtained in plasma samples are compared with values measured using either fluorescence polarization immunoassay or capillary gas chromatography-MS; a good correlation is observed between LC and both methods. The method has been applied in daily use to a large-scale study in a human healthy population, and some resulting data are discussed. (+info)